Abstract

Insulin resistance has long been recognized as a characteristic of aging. Many studies reported a beneficial role of oxytocin in obesity and type 2 diabetes. The aim of this study is to investigate the role of oxytocin in age-related insulin resistance in Wistar rats. Forty male rats were divided into four equal groups: young group, oxytocin-treated young group, old group and oxytocin treated old group. Oxytocin treated groups received Intraperitoneal (Ip) injection of oxytocin in a dose of 3 mg/kg body weight for 5 days. Experimental procedures included measurement of body weight, Body Mass Index (BMI) and intraperitoneal glucose tolerance tests. HOMA-IR index was also calculated. Assays of serum triglycerides, total cholesterol, LDL-C and HDL-C as well as serum IL-1β, IL-6 and TNF-α were performed. Malondialdehyde (MDA) and mRNA of IL-6, TNF-α and IL-1β were measured in Soleus Muscle (SM) and EF homogenates. Compared to young rats, old rats exhibited significant increase in BMI and HOMA-IR as well as an abnormal glucose tolerance test. Serum triglycerides, IL-6 and TNF-α were significantly increased in old rats. Furthermore, the MDA and mRNA of TNF-α and IL-6 of both SM and EF homogenates were significantly higher in old rats compared to young controls. These results, however, were notably lower in oxytocin supplemented rats compared to non-treated age matched group. In conclusion, oxytocin improves age related insulin resistance by alleviating inflammation and oxidative status in insulin sensitive tissues without affecting body weight.

Insulin resistance has been long recognized as a characteristic
of aging in humans and rodents [3]. Hence, aging has been
considered as one of the factors which accelerate the
development and progression of metabolic syndrome [4].
Moreover, insulin resistance could increase with age in relation
to several well-known age-related changes, including hormonal
changes, increased oxidative stress and inflammation [5].
Chronic, low-grade, systemic inflammation is widely accepted
as a significant risk factor underlying aging and age-related
type 2 diabetes [6,7]. The increased production of Proinflammatory
cytokines was shown to act in an autocrine or
paracrine manner to induce insulin resistance in peripheral
tissues and macrophages [8,9]. Coincidently, ROS generation as a result of oxidative stress has been implicated in the
development of insulin resistance and type 2 diabetes [10].

Apart from the traditional role of oxytocin in the physiology of
reproduction and lactation, the role of oxytocin in metabolic
regulation started to attract attention. Oxytocin-deficient mice
exhibited decreased insulin sensitivity and impaired glucose
tolerance [11]. Oxytocin was found to reverse obesity as well
as the related glucose and insulin disorders in mouse models
with therapeutic significance in the treatment of obesity and
insulin resistance. Oxytocin administration reduced obesity
related diabetic changes ranging from insulin resistance,
glucose intolerance, pancreatic islet hypertrophy and hepatic
steatosis [12,13]. The anti-inflammatory and antioxidant
effects of oxytocin were also reported to ameliorates the
immediate myocardial injury in heart transplant through downregulation
of the myocardial inflammatory response, reactive
oxygen species, and neutrophil-dependant myocardial
apoptosis [14].

Despite the metabolic advantage of oxytocin in increasing
insulin sensitivity and combating insulin resistance, together
with the reports about age related decline of oxytocin activity
at least in rats [15], no thorough studies have been reported from the perspective of its possible effects in these conditions
in aged rats. We postulate that oxytocin administration might
improve the age-associated insulin resistance and enhance
sensitivity of insulin dependent tissues. We also assume that
oxytocin might be able to lower inflammatory and oxidative
states of the insulin-sensitive tissues in aged rats. Hence, the
aim of this study was to investigate the effects of oxytocin on
age related insulin resistance in old rats. Moreover, we tested
the anti-inflammatory and antioxidant effects of oxytocin as a
possible mechanism for its action.

Materials and Methods

This randomized controlled study was conducted in the
Physiology Department, Faculty of Medicine, Ain Shams
University, Cairo, Egypt, from June 1 to August 31, 2015, and
was approved by the ethics committee of FMSU REC, Cairo,
Egypt. The work was undertaken on 40 male Wistar rats. The
rats were maintained under standard conditions of boarding.
The investigation conformed to the guide for the care and use
of laboratory animals published by the United States National
Institutes of Health. The rats were fed standard diet prepared in
our laboratory according to the normal nutritional dietary
requirements.

Rats were weighed and divided into four equal group (n=10).
Group I: young age group (2-3 Months old rats,), group II: old
age group (22-24 months old), group III Oxytocin treated
young rats, and finally group IV, oxytocin treated old rats.
Oxytocin treated groups received intraperitoneal injection of
oxytocin in a dose of 3 mg/ kg body weight Ip for 5 days.
Control rats were injected with saline as a vehicle. The dose of
oxytocin was chosen according to a pilot study in our
laboratory and previous studies [13]. No known side effects of
oxytocin were reported in the literature and oxytocin
administration was considered safe with no reliable side effects
for short term use in controlled research settings [16].

Experimental procedures

Glucose tolerance tests were conducted on overnight fasted
rats. A blood sample was taken from the tail of each rat and
was used to determine blood glucose and serum insulin levels.
Rats were then given a bolus injection of glucose (2 g/kg) into
the intra-peritoneal cavity. Blood glucose concentrations were
measured in other samples from the rat's tail with a glucometer
at 30, 60, 90 and 120 min. Insulin was measured using a rat
insulin enzyme-linked immunosorbent assay kit (ALPCO
Diagnostics Salem, NH). HOMA-IR test was used as a marker
for insulin resistance using the equation previously described
[17]. HOMA-IR=(fasting glucose × fasting insulin)/22.5.
Insulin concentration is expressed in μU/L and glucose in
mmol/L.

Rats were then weighed and anaesthetized with thiopental
sodium 40 mg/kg intraperitoneally (Sandoz GmbH, Kundl-
Austria). Body lengths were measured for later calculation of
BMI using the formula: weight in kg/ body length in m2 [18].
Blood samples were collected from the abdominal aorta, centrifuged, and the serum was stored at -20°C in separate
aliquots for biochemical assays. From each rat the soleus
muscle of one leg was carefully dissected from the surrounding
tissue, stored at -80°C for determination of MDA, and for
cytokine mRNA measurement. Epididymal pads of fat were
removed, stored at -80°C for measurement of MDA and
mRNA expressions of pro-inflammatory cytokines IL-1β, IL-6
and TNF-ɑ.

Biochemical analysis

The following analyses were carried out for cholesterol,
triglycerides, and High Density Lipoprotein (HDL) using kits
from Biodiagnostic, Egypt: low density lipoprotein (LDL) was
calculated according to the formula described elsewhere [19].
The serum concentrations of IL-1β, IL-6 and TNF-α were
determined by ELISA (Life Technologies, USA) using DS2
automated ELISA analyser (Dynex Technologies,USA),
according to the manufacturer’s instructions.

Measurement of tissue MDA

Soleus muscles were homogenized in cold KCl solution
(1.5%); MDA levels were estimated by the double heating
method [20]. The same steps were repeated to determine the
MDA in adipose tissue.

Expression of mRNAs of genes in the soleus muscle
and epididymal fat

RNA was extracted from the homogenized soleus muscle and
epididymal fat. Real time quantitative fluorescence PCR with
SYBR Green was used to measure expression of TNF-α, IL-1
and IL-6 mRNAs in the soleus muscle and epididymal fat with
GAPDH as an internal reference. The gene sequences were
identified in Gene Bank for the design of specific primers
(Table 1). Total RNA was extracted using Trizol (Invitrogen,
USA) according to the manufacturer’s instructions. Then, 4 μl
of total RNA were subjected to reverse transcription with
random primers, and M-MuLV reverse transcriptase
(Fermentas, #EP0451, European Union) in 20 μl of reaction
mixture at 37°C for 1 h and then at 95°C for 3 min using a
PCR instrument, to convert RNA into complementary DNA
(cDNA). Then, 5 μl of cDNA were added to the 50 μl reaction
mixture, followed by amplification in an automatic quantitative
fluorescent PCR instrument (Model 7500, Applied Biosystems,
USA). The PCR conditions were: pre-denaturation at 93°C for
3 min, 40 cycles of denaturation at 93°C for 30 sec, annealing
at 55°C for 45 sec, and extension at 72°C for 45 sec. All gene
expressions were normalized to GAPDH, which served as an
internal control for the quality of isolated RNA from each
homogenized muscle and adipose tissue samples. The data
represented relative quantification, using the comparative
delta-delta Ct method. The gene expression is reported as
relative quantities in subjects with or without cardiovascular disease as related to an external reference sample and normalised to an endogenous reference.

In young animals, no significant changes in oxytocin treated
young rats as compared to young control regarding all the
parameters studied. Both the initial and final body weights
were significantly higher in control and oxytocin-treated old
rats compared to control and oxytocin-treated young animals
respectively (P˂0.001 each). Moreover, the body weights at the
end of the experimental period in all groups studied were not
significantly different from their corresponding initial values.
In the old groups, non-treated rats showed significantly higher
BMI and HOMA-IR as compared to young controls (P˂0.05)
while their fasted blood glucose and serum insulin values were
not significantly different. However, blood glucose levels
during IP glucose tolerance test at 0.5, 1 h, 1.5 h and 2 hours
after glucose challenge were significantly higher in old rats
compared to young control rats (P<0.001). In contrast,
oxytocin-treated old rats showed significantly lower HOMAIR
index (P˂0.05) when compared to non-treated old rats. In
addition, blood glucose level during IP glucose tolerance test
showed significant decline in oxytocin treated old rats
compared to their age matched control at time 0.5, 1, 1.5 and 2
hours (P< 0.001) as shown in Tables 2-4).

Young

Young OXY

Old

Old OXY

Starting BW

108.5+1.32

110+1.33

319*+2.01

321.8#+2.08

Final BW

112.2+1.44

113.2+1.56

322.1*+2.24

328.9#+2.01

The change

2.7+0.45

3.2+0.78

3.1+0.46

7.1+0.12

*P˂0.001 significant from young group; #P˂0.001 significant from young Oxy group.

Table 2. Mean+SEM of starting and final as well as the changes of
body weight (gm) in control and oxytocin-treated young and old rats
during the 5-days treatment period.

Young

Young OXY

Old

Old OXY

BMI

4.55+0.04

4.53+0.04

5.42*+0.04

5.26+0.04

Fasting blood glucose
(mg/dl)

80.5+0.94

79.7+0.78

84.5+0.53

81.4+0.48

Fasting insulin
(µU/ml)

7+0.71

7.1+0.77

8.11+0.14

7.3+0.10

HOMA-IR

1.39+0.02

1.4+0.02

1.69*+0.03

1.46#+0.02

*P˂0.05 significant from young group; #P˂0.05 significant from old group.

*P˂0.001 significant from young group; #P˂0.001 significant from old group.

Table 4. Blood glucose during glucose tolerance test.

Compared to young controls, old rats showed significantly
higher serum levels of triglycerides, IL-1β, IL-6 and TNF-α
(P˂0.001, P˂0.05, P˂0.05 and P˂0.01, respectively). However, the changes in serum LDL-C, HDL-C and total cholesterol
were statistically insignificant. On the contrary, oxytocintreated
old animals exhibited significantly lower serum levels
of triglycerides as compared to age matched controls (P˂0.05).
Similarly, IL-1β, IL- 6 and TNF-α were significantly lower
(P˂0.05, P˂0.05, P˂0.01 respectively). However, the changes
in LDL-C, HDL-C and total cholesterol were not significantly
different in these groups (Table 5).

Young

Young OXY

Old

Old OXY

Triglycerides (mg/dl)

48.4±0.26

49.3±0.58

56.6***±0.64

51.2#±0.38

LDL-C (mg/dl)

57.82±1.41

39.34±0.19

47.2±1.73

40.5±1.25

HDL-C (mg/dl)

46.5±0.74

47.9±0.77

42.5±0.72

46.7±0.72

Cholesterol (mg/dl)

94±1.05

97±1.75

101±1.35

97.4±1.21

Plasma IL-1β (pg/ml)

2.1±0.1

2.12±0.06

3.42*±0.09

2.60#±0.08

IL-6 (pg/ml)

2,13±0.13

2.24±0.13

3.99*±0.10

2.88#±0.07

TNF-α (pg/ml)

3.22±0.06

3.16±0.07

5.68 **±0.19

3.48##±0.14

*P˂0.05, **P˂0.01, ***P˂0.001 significant from young group, #P˂0.05, ##P˂0.01 significant from old group.

Table 5. Effect of Oxytocin (OXY) on the serum lipids (triglycerides, total cholesterol, LDL-C, HDL-C) and pro-inflammatory cytokines (IL-Iβ,
IL-6 and TNF-α in young and old rats (mean+standard error of the mean, n=10).

Old rats showed significantly higher MDA levels of soleus
muscle and epididymal fat homogenates when compared with
young controls (P˂0.05 and P˂0.01, respectively). Moreover,
soleus muscle homogenates of old rats showed significantly
higher mRNA of IL-1β and TNF-α compared to the young
group (P˂0.05 each). Similarly, significantly higher IL-6 and
TNF-α (P˂0.05 each) were found in epididymal fat
homogenates of old rats. However, the higher levels of mRNA
of IL-6 of soleus muscle and IL-1β of epididymal fat of old
rats did not reach the level of significance when compared with
the corresponding values of young controls (Table 6).

Young

Young OXY

Old

Old OXY

SM homogenates

MDA

17.1±0.27

16.6±0.30

21.3*±0.31

18.0#±0.21

IL-1β

1.34±0.07

1.26±0.07

2.13*±0.06

1.42##±0.02

IL-6

1.13±0.04

1.16±0.03

1.43±0.04

1.26±0.04

TNF-α

1.24±0.04

1.21±0.04

1.74*±0.04

1.38#±0.02

EF homogenates

MDA

15.6±0.27

14.7±0.28

20.5**±0.37

16.3##±0.22

IL-1β

1.62±0.05

1.55±0.08

2.01±0.05

1.73±0.08

IL-6

1.40±0.07

1.31±0.05

2.15*±0.04

1.24###±0.06

TNF-α

1.21±0.06

1.33±0.05

1.94*±0.10

1.19#±0.07

*P˂0.05 ***P˂0.001 significant from young group, #P˂0.05 ##P˂0.01 significant from old group.

Table 6. Effect of oxytocin treatment on tissue content of MDA and
mRNA of IL-1β, PL-6 and TNF-α in young and old rats (mean+SEM
n=10).

Expressing our data as Δ Ct oxytocin administration to old rats
resulted in significantly lower soleus muscle content of MDA
and mRNA of IL-1β and TNF-α as compared to young
counterparts (P˂0.05, P˂0.001, and P˂0.05, respectively).
Similar significant reduction in epididymal fat content of MDA
and mRNA of IL-1β and TNF-α was found with oxytocin
supplementation (P˂0.01, P˂0.001 and P˂0.05, respectively).
However the mRNA of IL-6 of soleus muscle and IL-1β of
epididymal fat did not differ significantly between oxytocintreated
and untreated old rats (Table 6).

Table 7. Correlation coefficient (r) between HOMA-IR index and both
the inflammatory and oxidative markers in all rat groups.

Discussion

The present study demonstrated age related decline in glucose
tolerance reflected in an abnormal glucose tolerance test and
significant elevation of HOMA-IR index. There were also
significant increases in tissue and plasma content of proinflammatory
cytokines and thiobarbituric acid reactive
substances. The apparent insulin resistance and the associated
inflammatory and oxidative patterns were improved in old rats
upon systemic oxytocin administration.

The deterioration of insulin homeostasis in aged rats agrees
with the previous studies [5,21]. The improvement of age
related glucose tolerance upon oxytocin administration
indicates involvement of oxytocin in peripheral metabolic
functions of skeletal muscle and adipose tissue. Oxytocin
enhancement of glucose disposal evidenced by glucose
tolerance test reflects improved insulin sensitivity of oxytocin supplemented old rats. These data are in accordance with
others [22], and confirmed by the decrease in glucose tolerance
and insulin sensitivity in oxytocin deficient mice [11]. On the
other hand, contradicting reports about the effect of oxytocin
may be attributed to the differences in the dose of oxytocin,
animal species and the experimental model used in their study
[23].

The effect of oxytocin on glucose tolerance and insulin
resistance was previously attributed to the body weight
lowering and antiobesity effect [22]. Surprisingly, we did not
find any significant differences in the body weight or body
mass index when oxytocin treated rats compared with age
matched non-treated counterparts. The lack of body weight
lowering effect was supported by other studies where
exogenous oxytocin administration was not associated with
significant changes in body weight of atherosclerotic mice [24]
or hyperlipedimic rabbits [25] In addition, oxytocin showed its
therapeutic effect against prediabetic or diabetic animal model
regardless its effect on obesity [13]. Thus we could attribute
the beneficial effect of oxytocin in aged rats in our study to an
anti-obesity-independent mechanism. One of the mechanisms
by which aging contributes to insulin resistance is chronic
inflammation where accumulation of pro-inflammatory
cytokines had a negative impact on insulin signaling during
aging [5].

Our data showed significant elevation of proinflamatory
cytokines both in plasma and in insulin sensitive tissues. We
could thus attribute the state of age related insulin resistance to
the state of chronic inflammation displayed in old rats. These
inflammatory cytokines act in autocrine and paracrine manner
to induce insulin resistance in peripheral tissue. Moreover, our
results showed a highly significant positive correlation
between insulin resistance index (HOMA-IR) and
inflammatory markers, either their systemic concentration or
their level of gene expression in adipose tissue or in the muscle
(Table 7).

The beneficial effect of oxytocin could thus be related to the
improvement of the negative impact of pro-inflammatory
cytokines on insulin signaling during aging. Our data showed
down regulation of the expression of pro-inflammatory genes
in epididymal fat and skeletal muscle with oxytocin
administration. Although our assumption regarding this
molecular mechanism of oxytocin in insulin resistance is
totally new, this view is supported by the findings in other
similar situations. Previous reports had showed that oxytocin
infusion in models of myocardial infarction improved function
in the injured heart through reduction of inflammation [26,27].
Furthermore, chronic peripheral oxytocin administration
inhibited inflammation and atherosclerotic lesion development
where expression of adipokines from visceral adipose tissue
was indicative of decreased adipose tissue inflammation [25].
Specifically, oxytocin infusion reduced the secretion of IL-6
from epidymal fat ex-vivo in APOE -/- mice [24].

We assume that TNF-α and other pro-inflammatory cytokines
in could be the link between inflammation, aging and IR. The
increased TNF-α stimulated inhibitory phosphorylation of serine residues of IRS-1 either directly [28] or through
activation of IKKβ [29] or JNK [30] pathways. Specifically,
phosphorylation of these residues impedes the normal
association of IRS-1 with insulin receptors, thereby impairing
the downstream of insulin signaling [31,32]. We could also
extrapolate that the anti-inflammatory effect of oxytocin could
disturb the IKKβ and JNK pathways inhibiting serine
phosphorylation of IRS-1 and improving insulin signaling.
Inhibition of JNK or IKKK [30,33] improved insulin
sensitivity in various models of insulin resistance in association
with reduced inhibitory serine phosphorylation of IRS-1 [30].

Furthermore, we have demonstrated a significant age-related
increase in MDA reflecting increased free radical production in
old rats, in accordance with previous studies [34]. The
significant positive correlations between MDA content of
epididymal fat and soleus muscle and HOMA-IR index in our
study provide an index about the close association between
reactive oxygen species and IR [35,36]. Initially, mutations and
deletions which occur in DNA lead to impaired function of the
respiratory chain and enhanced ROS production with
subsequent accumulation of fatty acid metabolites [37].
Eventually, activation of protein kinase C leads to activation of
IKK and JNK followed by impaired insulin signalling [38].

The significant decline of malondialdehyde content in insulin
sensitive tissue of oxytocin-treated old rats addresses the
antioxidant effect of oxytocin supplementation in combating
insulin resistance. This finding agrees with the recent
conclusions about the negative correlation between maternal
levels of oxytocin and both the total oxidative status and the
oxidative stress indices [39,40]. This amelioration of oxidative
status converges with the anti-inflammatory results in
inhibiting IKK and JNK pathways potentiating the insulin
signaling mechanism.

In summary, the present study demonstrates that oxytocin
supplementation attenuated the age-related inflammatory and
oxidative messier in insulin sensitive tissues independent of its
effects on body weight. It subsequently improved insulin
resistance state and thus may provide a possible therapeutic
intervention in the aging population.